The symbiotic association between legumes and nitrogen fixing rhizobia culminates in the formation of root nodules. Legumes control the extent of root nodule formation through a regulatory mechanism in which nodulating roots induce a shoot response that signals back to the roots, inhibiting further nodule initiation. Disruption of genes involved in this regulatory process lead to super or hypernodulation. The lss (like sunn supernodulator) mutant of Medicago truncatula has been identified as a naturally occurring supernodulation mutation in the Jemalong cultivar. The phenotype of lss is practically indistinguishable from the phenotype of the previously isolated sunn mutant with respect to root length, nodule number and shoot control of the phenotype. However, lss and sunn plants nodulate differently in the presence of nitrogen, endogeneous auxin (IAA) or ethylene. SUNN gene expression is also altered in lss shoots versus sunn shoots. F1 analysis of progeny from lss crossed with sunn demonstrated that the two mutations do not complement each other and the nodule and root phenotypes of the F1 are indistinguishable from either sunn or lss. Because, sequencing of the SUNN gene, the RLP1 gene and the region between them showed no mutations, the lss phenotype probably does not result from some lesion in this approximately 12 kb region. F2 plants from the lss/sunn cross segregated wild type plants in a ratio suggesting separate but linked genes, confirmed from mapping of the locus through a separate cross to the polymorphic ecotype A20. Analysis of segregating CAPS markers in the F2 from this cross determined that lss maps to an 800 kb area at the bottom of LG4, which also containssunn. Methylation analysis by bisulfite sequencing of the SUNN promoter and 3'UTR in shoots and roots of lss showed no significant differential methylation from wild type to account for the lss supernodulation phenotype.